1. Field of the Invention
The present invention relates to the acedan derivatives having an extended π bond, which are novel two-photon absorbing fluorescent compounds, a method of preparing the acedan derivatives, an application of the acedan derivatives for bioimaging and for two-photon microscopy imaging of amyloid-beta plaque in an animal model with Alzheimer's disease.
2. Discussion of Related Art
Bioimaging techniques based on a fluorescence signal are widely used as a method of visualizing not only an organelle but also tissue in an animal model. Among the techniques, the development of fluorescent probes expands the application area for specific material analysis and imaging in vivo.
Most fluorescent probes which have been reported up to now are based on one-photon absorption fluorophores, and undergo the imaging process in accordance with one-photon microscopy (OPM). However, the use of general one-photon microscopy has a disadvantage in tissue imaging in that the image quality is reduced due to strong light scattering and only shallow tissue at a depth of 100 μm or less may be imaged.
The use of a nonlinear optical microscope provides insensitivity to the above-described light scattering and properties suitable for high-resolution imaging. Two-photon microscopy (TPM) is one type of the nonlinear optical microscopy, which allows a fluorophore to be excited by simultaneous irradiation with two photons having energy with a wavelength corresponding to a half of the one photon which is used in one-photon microscopy (OPM). Furthermore, a two-photon fluorophore is only excited at a focal point, and thus an image with high resolution may be implemented. Accordingly, the use of two-photon microscopy has advantages for its application in bioimaging such as high tissue permeability, an increased photon-penetrating depth, low photo-damage to bio-tissue, low photo-bleaching, etc. Further, the interference effect from autofluorescent materials in vivo is small.
The number of two-photon absorbing (TPA) fluorophores having optimal characteristics required for bioimaging is limited. As a representative two-photon absorbing fluorophore, acedan (1-(6-dimethylaminonaphthalene-2-yl) ethanone) represented by the following Formula 10 is a dipolar dye having a D-π-A structure in which an electron donor (D) and an electron acceptor (A) are included in an aromatic ring (π-system), and has a high photostability and large two-photon absorption cross-section value, and thus is used in the two-photon microscopy imaging study for a living cell and tissue.

However, acedan has a shorter maximum absorption wavelength (<400 nm) and maximum emission wavelength (<550 nm), and thus has a disadvantage in that acedan is highly affected by the autofluorescence of fluorophores in vivo such as tryptophan, tyrosine, phenylalanine, retinol, riboflavin and nicotinamide adenine dinucleotide, etc.
In addition, the general acedan derivatives have a problem in that a low-power laser is used, or a process of removing an autofluorescent signal by signal processing is further needed so as to avoid the effect from autofluorescence and obtain a clear two-photon fluorescence microscope image. Accordingly, in order to avoid the effect from autofluorescence and obtain a clear two-photon fluorescence microscope image, novel two-photon absorbing fluorophores which are capable of being excited at a longer wavelength of about 900 nm such that the fluorophores are suitable for biological optical windows should be ensured.
As another example of the acedan derivative, GCTPOC represented by the following Formula 2 is a dipolar dye having a D-π-A structure, which is based on a green fluorescent protein chromophore (GFP chromophore, p-HOBDI) represented by the following Formula 3. However, the above-described fluorophore also shows a maximum absorption band in a short wavelength area (<400 nm) like acedan, and thus has a problem in that the autofluorescence due to a fluorophore in vivo is shown at the two-photon excitation condition (<800 nm).
